Theses - Clinical Neurosciences
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Item Open Access Selective Degradation of Aggregated Tau Protein Via Vectored Nanobody-TRIM21 RING Fusion ConstructsBenn, JonathanThe accumulation of intraneuronal aggregated tau protein underlies many common neurodegenerative diseases and is a key target in therapeutic development. Whilst promising in pre-clinical models, tau targeting antibodies have performed poorly in clinical trials, potentially due to low target engagement. Such antibodies rarely access the intracellular domain where the majority of tau aggregates reside. However, recent findings indicate that extracellular tau:antibody complexes can be internalised, whereupon they are bound and degraded by the intracellular Fc receptor and E3 ubiquitin ligase TRIM21. It was therefore hypothesised that intracellular aggregated tau could be targeted for degradation by exploiting this TRIM21 pathway. This thesis describes the development of TRIMinators, vectored constructs designed from the minimal antibody and TRIM21 domains required for the intracellular degradation of aggregated tau. These TRIMinators consist of an anti-tau nanobody fused to the E3 ubiquitin ligase domain of TRIM21. This E3 ligase activity is initiated by intermolecular clustering, which was hypothesised to allow for the selective degradation of multivalent aggregated tau:TRIMinator complexes whilst leaving physiological monomeric tau unaffected. In cell line models, TRIMinator expression protects against seeded tau aggregation and leads to the rapid and selective degradation of pre-existing aggregates, leaving monomeric tau unaffected and reducing the levels of seed competent tau species. This activity is dependent on the ubiquitin proteasome system (UPS), with the selective degradation of aggregated tau over monomeric species being determined by the affinity of the anti-tau nanobody. Delivered via adeno-associated viral (AAV) vectors, TRIMinator expression ablates seeded tau aggregation in primary neuronal models and reduces levels of tau pathology in aged transgenic P301S tau mice ten days after administration. The TRIMinator constructs described in this thesis are not only capable of inhibiting the aggregation of tau protein, but also remove pre-formed aggregates whilst leaving physiological monomeric tau unaffected. This system of selective aggregate degradation could be exploited in the development of future therapeutics not only for tauopathies, but for the various other neurodegenerative proteinopathies driven by pathological protein aggregation.Item Open Access Magnetic Resonance Based Imaging for Degenerative Cervical MyelopathyDavies, Benjamin; Davies, Benjamin [0000-0003-0591-5069]Degenerative cervical myelopathy [DCM] is a progressive, spinal cord injury, estimated to affect 1 in 50 adults. Spinal cord compression from degenerative changes of the cervical spine, such as intervertebral disc prolapse, osteophyte formation or ligament thickening have been considered the disease hallmark. Assessment of spinal cord compression using Magnetic Resonance [MRI] has therefore become a gold standard assessment for diagnosis and popular outcome assessment. This approach has limitations, which are poorly recognised in clinical care and research, representing a knowledge translation gap. Improving MRI assessment of DCM is therefore a recognised research priority. A range of different approaches are at various stages of development and may offer improved assessments of DCM. Multi-parametric quantitative MRI, including diffusion-based imaging, is one prominent example. Structural MRI, the sequences routinely used in clinical care today, too may offer additional and/or more readily accessible information. For example, the nature and interaction of degenerative pathology, or in combination with finite element analysis. This thesis tests the hypothesis, “the application and/or interpretation of MRI can be optimised to improve the diagnosis, monitoring and/or surgical decision making of Degenerative Cervical Myelopathy” using a variety of research methodologies. Initially using systematic review and consensus, tools are developed to support knowledge translation around structural MRI today. Then the practicalities, validity, and value of implementing multi-parametric quantitative MRI are considered using a cohort of patients undergoing surgery and a targeted protocol. Then it explores the clinical significance of the presence or absence of degenerative pathology to the diagnosis of DCM. Finally, it explores a role for finite-element analysis to extend MRI, through a systematic review and development of a computational model. This research therefore creates recommendations for current practice, whilst exploring new imaging solutions for DCM. Overall, its findings support the shifting focus of DCM as a disease of spinal cord compression to instead a disease defined by mechanical stress, vulnerability, and time with injury.Item Embargo Master regulators of neuronal maturation in NGN2 reprogrammed iPSC-derived iNeuronsPatili, EleniNeuronal maturation is an intricate and tightly-controlled process in neurodevelopment, during which neuronal cells acquire their mature molecular, morphological and electrophysiological properties to function as the central components of the nervous system. However, the cell-intrinsic mechanisms and transcriptional programs that govern it remain poorly understood. To address this gap in knowledge, the objective of this thesis was to identify the transcription factors (TFs) that control neuronal maturation and elucidate the pathways through which they exert their function. The present work takes advantage of the recent discovery that Neurogenin 2 (NGN2) overexpression in induced pluripotent stem cells results in their rapid conversion into excitatory induced neurons (iNeurons) that are morphologically complex and electrophysiologically active after 2 weeks in culture. In addition, the inducible control of NGN2 expression from genomic safe harbour sites has provided a deterministic model system, enabling the generation of pure neuronal cultures within days, with minimal variability. Culturing iNeurons together with primary rat glia results in more mature networks with early synchronous burst activity, as measured on multi-electrode arrays (MEAs). Candidate master-regulators of iNeuron maturation were identified using TF motif prediction tools and a dataset combining ATAC-seq and RNA-seq analysis of a granular time-course experiment during NGN2 reprogramming, in the presence and absence of glia. Predicted TFs were found to also be expressed during human cortical development and mutations in them are enriched in neurodevelopmental disorders, suggesting potential functional roles *in vivo*. To simultaneously investigate the function of 25 potential master-regulator TFs, I conducted a pooled CRISPR knock-out (KO) screen with single-cell transcriptome readout (CROP-seq screen) in iNeurons cultured with glia. Differential expression analysis implicated TFs in cellular processes hypothesized to promote neuronal maturation, including autophagy, oxidative stress response, cellular respiration and lipid metabolism. To assess the effects of transcription factor KO on the maturity of iNeurons, single-cell RNA-sequencing reads were analysed with a composite metric, namely neuronal maturity index, which suggested incomplete transcriptome transition towards mature states for a number of gene KOs. I next generated KO lines for the transcription factors CUX2, ZNF384, FOSL2, PATZ1 and ONECUT2 for further in-depth investigation. MEA analysis demonstrated notable impairments in iNeuron electrophysiology, while neurite and synaptic connectivity profiling revealed stunted neurite outgrowth and synapse maturation, respectively. Lastly, the investigation of cell metabolism revealed that PATZ1-KO iNeurons displayed decreased cellular respiration, ROS levels and autophagic activity, which may be directly linked to the maturation deficits. In conclusion, the present work highlights the importance of transcriptional regulation in the process of neuronal maturation. It identified and confirmed the functional role of a number of transcription factors, enabling not only a mechanistic understanding of neuronal maturation but also providing a basis for further research in neurodevelopmental conditions.Item Open Access Computational Models Explaining Cochlear Implant Principles: A Hypothesis, Applications and Physical ValidationsHuang, BotianThis thesis combines computational modelling with cadaveric and clinical data to deepen our understanding of cochlear implants (CIs). For the past two or three decades, improvements in CI performance have been limited due to gaps in our understanding of how CI interacts with auditory nerves. Persistent questions, such as the unexplained polarity effect and the impact of CI-modiolus distance, have remained unclear. This research aims to improve our knowledge of CI principles. It introduces a hypothesis, based on precise computational models, that explains these unclear phenomena, suggesting the internal auditory meatus (IAM) as a key factor in neural activations by CI. This work also involves validating these computational models through physical experiments on cochleae and CIs. It compares clinical measurements from patients and from simulations like extra-cochlear electrodes and scalp voltages. Additionally, it proposes a proof-of-concept in-vitro cell culture model based on these simulations. In detail, Chapter 2 focuses on validating these models against human temporal bone specimens for evaluating the accuracy of computational methods. Chapter 3 develops a comprehensive head model that sheds light on how CIs affect neural pathways and electric field intensities, with a special focus on the IAM. This leads to the proposed hypothesis. Chapter 4 explores clinical applications, studying extra-cochlear electrodes and simulating CI-induced scalp voltages, supported by cadaveric and clinical data. Chapter 5 introduces a novel in-vitro model for studying responses of spiral ganglion neurons (SGNs) to CIs. Overall, this thesis aims to advance our understanding of CI principles and opens up new possibilities for research in the field of auditory prosthetics.Item Embargo Investigating the TrkB Interactome during Development and in NeurodegenerationZbiegly, JuliaBrain-derived neurotrophic factor (BDNF) is a cardinal regulator of neuronal development, survival and synaptic plasticity, signalling via its major receptor, Tyrosine receptor kinase B (TrkB). In the developing brain, BDNF acts as a guidance cue influencing the direction of the axonal growth cone. In the mature brain, it ensures survival of neurons and synaptic plasticity. Guidance cues remodel the nascent local proteome in the developing nervous system. Recently, guidance cue receptors DCC, Neuropilin-1 and Robo2 were shown to interact with the translation machinery. Both the protein and mRNA interactions of these receptors help them to define which proteins are locally synthesized in response to stimulation. BDNF likewise regulates the local nascent proteome with high spatiotemporal resolution via TrkB activation. While there is extensive data on expression level, trafficking and signalling of TrkB, little is known about its protein and RNA interactions. We hypothesised that like DCC, Neuropilin-1 and Robo-2, TrkB shows similar interaction with the translation machinery. Immunoprecipitation studies for TrkB were performed and protein interactors were analysed by LC-MS/MS, interacting mRNAs were sequenced. A specific set of ribosomal proteins, RNA-binding proteins, rRNAs and mRNAs interacting with TrkB were detected in the embryonic mouse brain. However, in the adult mouse hippocampus, where BDNF-induced local protein synthesis mediates synaptic plasticity, no receptor-ribosome interaction was detected. This suggests a remodelling of the TrkB interactome during development and that BDNF modulates the nascent proteome in adult brain via different mechanisms. To elucidate possible new roles and regulatory mechanisms of TrkB in the mature brain, we screened for novel protein interactions in adult mouse hippocampus and cerebral synaptosomes. The TrkB protein interactome was largely composed of cytoskeleton and transport vesicle-related proteins, as well as transmembrane transporters. At the synapse, in particular, small molecule transporters interacted with TrkB, including ion and glutamate transporting excitatory amino acid transporter 1 (EAAT1). BDNF stimulation interrupts the TrkB-EAAT1 interaction and leads to increased EAAT1-dependent glutamate uptake in primary hippocampal cultures, elucidating a novel crosstalk between neurotrophin receptor and glutamate transporter. The BDNF-TrkB axis is impaired in ageing and in neurodegeneration. We have poor understanding of the processes occurring in these conditions. We hypothesised that the TrkB interactome may vary in aging and neurodegenerative disease as a possible contributor to their impaired signalling. Interestingly, a screen of the interactomes in the mouse brain showed that major TrkB co-precipitating proteins are not affected by ageing. In murine prion disease, which results in extensive neurodegeneration, a TrkB-interactome screen revealed increased association of a distinct set of cell-adhesion proteins with TrkB, including Connexin-43, a gap-junction adhesion molecule which is associated with other neurodegenerative diseases. The identification of novel disease-related interactions may yield future insights into dysregulated BDNF signalling in neurodegeneration. In conclusion, in this thesis, the interactome of TrkB was studied to increase our understanding of BDNF function in health and disease in the mouse brain. Novel interaction partners of the neurotrophin receptor TrkB were identified, during development, in adulthood and ageing, as well as in prion neurodegeneration. Specific interactors identified may, upon further investigation, represent novel mechanistic insights into BDNF signalling and possibly new targets to tackle dysregulated neurotrophic signalling in many pathological conditions.Item Open Access From Acute Injury to Chronic Disease: Exploring the Neurological Consequences of Mild Traumatic Brain Injury through Functional NeuroimagingWoodrow, Rebecca; Woodrow, Rebecca [0000-0001-8097-5905]Traumatic brain injury (TBI) is a global health crisis, with incidence rates growing rapidly. Further, TBI is the leading cause of injury-related death and disability, with lifelong impacts on the individual and their families. Of increasing concern is mild TBI (mTBI), which is overexpressed in the population yet lacks adequate attention in current clinical practice. Despite many experiencing long-term consequences of so-called ‘mild’ TBI, the relative paucity of clinical understanding and care of these patients has created a disconnect between injury and outcome. Thus, we are currently unable to explain the neurological underpinnings of poor outcome after mTBI, predict who might experience long-term effects, or sufficiently treat these patients. In this thesis, I aim to re-frame ‘mild’ TBI as a non-trivial and long-term disease, using multiple neuroimaging methods to better understand and prognosticate the outcomes of these individuals. Using data from collaborative multi-centre project CENTER-TBI, Chapters 2-5 explore the acute and enduring neurological effects after even the ‘mildest’ TBI. Expressly, those individuals within hospital settings who do not present existing markers of poor outcome such as damage on computerised tomography (CT) or pre-injury neuropsychiatric conditions. Even with such ‘mild’ injury, Chapter 2 finds that this does not necessitate mild outcome, as 47% of our mTBI group show incomplete functional and/or symptomatic recovery at 6 months post-injury. This chapter further identifies that common structural neuroimaging methods or blood-based biomarkers are not associated with poor outcome in this cohort, necessitating the need for novel markers of chronic outcome. Chapter 3 establishes mTBI as a global functional disorder, using resting-state functional magnetic resonance imaging. Explicitly, all resting-state networks intrinsic to healthy brain function show vast alterations in functional connectivity. Additionally, these networks show injury-induced changes in how they are spatially distributed across the brain using a novel measure of component distribution complexity. Both measures show preliminary associations between disrupted network functional connectivity and poor outcome, but require further acute biomarkers to successfully differentiate chronic outcome. Chapter 4 begins to focus investigations on a globally connected subcortical structure, previously ignored in TBI; the thalamus. In the same cohort of mTBI, I find acute thalamic hyperconnectivity, with specific vulnerabilities of individual thalamic nuclei. These acute fMRI markers differentiate those with versus without chronic post-concussive symptoms, additionally with time- and outcome-dependent relationships in a sub-cohort followed longitudinally. Moreover, chronic emotional and cognitive symptoms are associated with acute changes in thalamic functional connectivity to known serotonergic and noradrenergic targets, respectively. This begins to bridge the gap between macrostructural and microstructural investigation; translating findings from acute imaging into treatment-relevant targets and aiming for each field to mutually influence the other for therapeutic development. Chapter 5 additionally finds that thalamocortical connectivity is exacerbated in the special interest group of repeat mTBI. These results further establish thalamic pathophysiology as a marker of acute injury and outcome, and has important implications for both public and professional sports players. In the final experimental Chapter 6, I further explore the evolving and potentially lifelong thalamic neuronal consequences of TBI, across all severities. Using rarely-collected 11C-flumazenil positron emission tomography (PET), the thalamus shows unique markers of selective neuronal loss extending many years post-injury, which additionally mirror regions of cortical damage. These thalamic markers are related to multiple adverse outcomes, thereby substantiating that the thalamus can link the injury event with the long-term disease of TBI. Overall, this thesis establishes functional neuroimaging as an invaluable tool for better understanding and prognosticating mTBI. Moreover, I propose the thalamus is a common source of injury, outcome, and long-term disease following TBI. It thus demands greater recognition and investigation in the TBI community, which is beginning to take flight. So-called ‘mild’ TBI is neither trivial nor temporary, and has traditionally been dismissed in public and clinical settings. For the many individuals experiencing long-term symptoms, multidisciplinary teams must work together to form new therapeutic pathways, towards a precision medicine approach. Only then will future research and healthcare professionals be able to sufficiently care for this growing population.Item Embargo Microglial succinate receptor 1 (SUCNR1) sustains chronic CNS inflammationKrzak, GrzegorzThe rapidly evolving field of immunometabolism has shed new light on new fundamental properties of metabolites, which can regulate inflammation via ligand-receptor signalling modalities. Among these, succinate signalling via succinate receptor 1 (SUCNR1) plays a key role in controlling myeloid cells responses in systemic inflammatory conditions. However, how the succinate-SUCNR1 axis regulates myeloid cell functions in the central nervous system (CNS) inflammatory disorders, such as multiple sclerosis (MS), is still largely unknown. Herein, I first investigated the role of SUCNR1 signalling in mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. I found that SUCNR1 expression increases in the inflamed mouse CNS, where it is mostly expressed by myeloid cells in chronic inflammatory lesions. Post mortem single-molecule fluorescence in situ hybridization (smFISH) and histopathology analysis of people with progressive MS confirmed a predominant expression of SUCNR1 in chronic active and active white matter lesions. When EAE was induced in a constitutive Sucnr1 knockout (Sucnr1-/-) mice, I found that Sucnr1-/- led to a significantly worsened disease outcome compared to controls, which was associated with increased peripheral immune infiltration and relatively lower numbers of microglia seen via ex vivo suspension mass cytometry (CyTOF) immunophenotyping. Mechanistically, I found that SUCNR1 has opposite effects on macrophages, where it attenuates their pro-inflammatory activation, compared to microglia, where it promotes the response to both pro-inflammatory stimuli and succinate stimulation in vitro. Concordantly, I found that the in vivo genetic perturbation of Sucnr1 function in microglia only was capable of attenuating EAE disease severity, ameliorating neuropathological outcomes, and promoting the acquisition of a preferential homeostatic microglial phenotype as seen via CyTOF and single-cell RNA sequencing (scRNAseq) analyses. In conclusion, my work clarified the role of SUCNR1 as metabolic receptor of myeloid cells that drives microglial persistent activation and identified a new pathway that could be leveraged to ameliorate persistent CNS inflammation in MS.Item Open Access hiPSC-Derived Cerebral Small Vessel Disease ModelsAl-Thani, MahaCerebral small vessel disease (cSVD) is an umbrella term of disorders that affect the small perforating arteries of the brain and is a major cause of stroke and dementia worldwide, but its pathology is yet to be known. We hypothesise that the integrity of the blood brain barrier (BBB) is disrupted and could contribute to increased BBB permeability. Evidence also shows a role of the extracellular matrix (ECM) in the progression of cSVD pathology. We generated *in-vitro* co-culture models using human induced pluripotent stem cells (hiPSCs) with COL4A1, COL4A2, and HTRA1 cSVD-related mutations to assess the shared role ECM plays in cSVD. These mutations induced tight junction abnormalities with Occludin discontinuity and Claudin-5 accumulation in hiPSCs-derived Brain Microvascular Endothelial Cells (iBMECs), which resulted in decreased TEER levels and increased permeability. They also induced apoptosis, migration defects, and transcriptome changes in hiPSCs-derived Mural Cells (iMCs). A co-culture system made of iBMECs and iMCs revealed that iMCs exert a detrimental paracrine effect on iBMECs barrier integrity. These models also expressed high levels of MMP- 14 and show an increase in MMP-2 and MMP-9 activity; inhibiting MMP activity with either doxycycline or shMMP-14 rescued both iBMECs and iMCs phenotypical changes. Our models revealed that iMCs secretome influences barrier integrity, and the shared ECM phenotype include increased MMP-14 levels and tight junction abnormalities, which confirm our hypothesis of barrier disruption leading to BBB leakage. Our rescue models also provide basis for MMP targeting as a therapeutic approach in cSVD.Item Open Access Digitally quantified neuropathological correlates of structural and functional imaging biomarkers in progressive supranuclear palsyPansuwan, TanradaNeuroimaging measures are increasingly useful as *in vivo* biomarkers for differential diagnosis in neurodegenerative tauopathies. However, the relationship between imaging changes and neuropathology requires more thorough validation and beyond Alzheimer’s disease. In tauopathies, tau is the key protein where specific pattern of tau aggregation and distribution can distinguish between different tauopathies. With this, progressive supranuclear palsy (PSP) is a prime disease for investigating the relationship between imaging changes and tau burden. Early studies have revealed that semi-quantitative pathological tau measures are predictive of *in vivo* atrophy but is less predictive of intrinsic functional connectivity. However, the semi-quantitative pathological measures used in these studies have limitations such that they are prone to inter-rater variability and may be insensitive to subtle pathological patterns. Therefore, in this thesis, I first attempted to develop an automated pipeline for quantifying total and tau positive cell density for neuronal and glial cells in *post mortem* samples. This promises to be a more objective, detailed, and scalable solution for pathological assessment. However, cell classification with high accuracy has proven to be very challenging; I then adapted the pipeline to only focus on quantifying tau type-specific aggregates in PSP. I used the pipeline to quantify PSP-related tau aggregates across cortical and subcortical regions and found a strong correspondence between the digitally quantified tau burden and the current consensus PSP staging scheme. I further assessed its clinicopathologic predictive power and found that total cortical tau and subcortical neurofibrillary densities correlate with clinical severity measured prior to death. Lastly, I assessed the relationship between both structural and functional imaging measures and *post mortem* tau type-specific density. I found that total tau density was associated with *in vivo* functional connectivity but not atrophy, suggesting that structural measure may be a less reliable marker of tau burden than functional measure in PSP. Overall, I have demonstrated that digitally quantified tau pathology can be a powerful tool to deepen our understanding of the role of tau for mechanistic studies and diagnosis in neurodegenerative tauopathies.Item Open Access Engineering hiPSCs to enhance neural progenitor transplantation in degenerative cervical myelopathyPoulin, NoahNeural progenitor cell transplantation has been widely explored in traumatic spinal cord injury (SCI) but has yet to be evaluated as a treatment for degenerative cervical myelopathy (DCM), a non-traumatic form of spinal cord injury. Importantly, the extent of astrogliosis and extracellular matrix remodelling that could limit or enhance neural progenitor cell (NPC) graft efficacy in DCM is unknown. The first aim of this project was to examine changes in the extracellular matrix in DCM. Immunohistochemistry of post-mortem patient tissue and a mouse model of DCM indicated astrogliosis and chondroitin sulfate proteoglycan accumulation, which could inhibit NPC integration. Meanwhile, bulk-transcriptomics from human post-mortem tissue revealed no upregulation of astrogliosis- or fibrosis-related genes in DCM. Second, the extent of endogenous remodelling of perineuronal extracellular matrix structures that could enable functional connectivity of NPC grafts – and which may underlie compensatory plasticity in DCM – is unknown. Immunohistochemical analysis of perineuronal nets (PNN) in murine models of DCM showed no significant changes in the number of PNN-positive neurons. Additionally, transcriptomic analysis of PNN-related gene expression from human tissue similarly showed no significant changes in DCM. Therefore, while diffuse astrogliosis and ECM deposition occurs in DCM, endogenous PNN remodelling may not underlie compensatory plasticity. Next, given the importance of PNNs in regulating synaptic plasticity, elucidating mechanisms of their development and remodeling through in-vitro models of human neurons would be useful. No such systems have been reported thus far and it remains unclear whether forward reprogrammed hiPSC-derived glutamatergic and GABAergic neurons contain PNNs. The second aim of this thesis was to determine the extent of PNN formation in forward reprogrammed neurons. Both NGN2 (glutamatergic) and ASCL1/DLX2 (GABAergic) lines displayed punctate, immature PNN structures immunopositive for WFA, which increased along with aggrecan expression over 28 days post-induction. Analysis of existing single-cell and bulk-transcriptomic datasets revealed maturity-dependent increases in neurocan expression, a transient increase in brevican, and no significant increases in aggrecan expression, suggesting that the lectican responsible for WFA immunopositivity in these cells is likely neurocan. Finally, inducible expression of GDNF and ChABC from transplanted NPCs in DCM has the potential to improve graft survival and integration, while providing neuroprotective and regenerative benefits for host cells. Therefore, a dual safe-harbour tetracycline-inducible control system was used to express GDNF and ChABC in human induced pluripotent stem cells (hiPSC) and hiPSC-derived neural progenitors. Immunoblotting, enzyme linked immunosorbent assays, and qPCR were used to measure expression of GDNF and ChABC in-vitro, along with functional assays for ChABC. These established dox-inducible expression and secretion of GDNF and functional ChABC at both the iPSC and NPC stages. This system enables robust regulatable expression of GDNF and ChABC in hiPSC-derived NPCs and provides a path towards further evaluation of engineered NPCs in pre-clinical models of DCM and SCI.Item Open Access Apathy after stroke: clinical characteristics, association with functional outcome and effect on carer burdenPallucca, ClaudiaApathy is a multidimensional syndrome that frequently presents in stroke survivors and is characterised by a loss in motivation and initiative, reduced social interactions, and neutral emotionality. Apathy affects cognitive functioning, everyday activity including social life, and functional recovery. Despite the prevalence of apathy among the sequelae of stroke, an understanding of this symptom trajectory and its effect on patient and carer’s quality of life needs to be clarified. The research presented in this thesis mainly focuses on a prospective longitudinal study conducted in three acute stroke services in the East of England, UK. The main goal of the study was to evaluate the prevalence of *post*-stroke apathy and its association with outcome, mood, and cognition. Findings show that overall apathy tends to increase over one year after stroke and that different groups of patients present with different symptom trajectories. Moreover, the study results show that *post*-stroke apathy presents with specific patterns of impaired dimensions, which may vary depending on the measurement technique used, including method of scoring. After analysing the relationship between apathy, disability, and quality of life, my findings suggest that depression, more than apathy, might play an important role in determining functional outcome and recovery. The investigation of neurobiological bases of apathy found an association with white matter pathology, reinforcing the idea that chronic ischaemia coupled with acute lesions might set up a cascade of events leading to apathy. A new study was then set up to investigate the effects of apathy on carers of patients with small vessel disease and found that apathy is associated with higher care burden and distress. The results of this study pave the way for targeted intervention approaches. Overall, this thesis suggests that apathy is a symptom or syndrome presenting in about 20% of stroke patients and affecting patient and carer lives alike. A comprehensive characterisation of apathy holds clinical relevance and encourages the further development of new apathy treatments.Item Embargo Axon Degeneration and the Schwann Cell Early Injury Response: A Study in Mouse and ZebrafishMutschler, Clara; Mutschler, Clara [0000-0003-2189-2389]After peripheral nervous system injury, axons degenerate through a process termed Wallerian degeneration and Schwann cells transform into a repair phenotype. Axon degeneration is regulated by a signalling pathway controlled by the pro-degenerative axon death molecule sterile-alpha and toll/interleukin 1 receptor motif containing protein 1 (SARM1). Meanwhile, Schwann cells activate a distinct transcriptional response, digest myelin using myelinophagy, attract macrophages, and support the survival of damaged neurons and their growth and guidance to their target. The early Schwann cell injury response prior to and around the timing of axon degeneration has, however, not been investigated in great detail, and the identity of an axonal injury signal that induces this Schwann cell injury response remains elusive. In order to investigate axon Schwann cell interactions in vitro, I developed a novel compartmentalised dissociated dorsal root ganglion neuron and Schwann cell coculture model. I show that in this model, Schwann cells are initially axo-protective, as their presence delays degeneration, irrespective of their myelination status. In later phases after injury, they are then axo-destructive and fragment and phagocytose axons. To further investigate the role of Schwann cells after injury, I then characterised an in vivo model of peripheral nervous system injury in larval zebrafish. I describe the rate of axon degeneration after laser axotomy of the peripheral lateral line nerve in wildtype, as well as in sarm1 mutant animals. I show a characteristic delay in axon degeneration in sarm1 mutant zebrafish, while their myelination is normal. I then performed cell specific reexpression experiments with human SARM1 and show that neuronal SARM1 is sufficient to rescue axon degeneration. In a more traditional mouse model of peripheral nervous system injury, I performed a bulk RNA sequencing study of the distal tibial nerve after cut injury at the sciatic notch at early timepoints after injury in both Wildtype and Sarm1 knockout mice. Previous studies have mainly focussed on late timepoints, but I show a much earlier induction of the Schwann cell injury response, prior to myelinated axon degeneration. I then further investigated the timing of unmyelinated axon degeneration and show that these degenerate before myelinated axons do, at timepoints that correspond to the induction of the early Schwann cell injury response. I further show that Schwann cells likely do not express SARM1 and are insensitive to SARM1 activation, suggesting the delayed degeneration in Sarm1 knockout mice is solely due to the axonal absence of Sarm1. Overall, this thesis details novel methods to investigate peripheral nervous system injury and provides novel insights into early events after injury | both in Schwann cells and axons. Results provide insights into Schwann cell axon interactions after injury, and highlight key differences between myelinated and unmyelinated axons that warrant further investigation.Item Controlled Access Derivation of next generation research models of Parkinson’s diseaseAnsari, RizwanParkinson's disease (PD) is the second most common neurodegenerative disorder following Alzheimer's disease. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra region of the midbrain and the presence of intracellular inclusions, notably Lewy bodies composed of the alpha-synuclein protein (encoded by the SNCA gene), in the surviving neurons. The precise molecular and cellular mechanisms governing PD's onset, progression, and pathology remain incompletely understood. The development of advanced research models is pivotal in unravelling the mysteries surrounding PD's pathogenesis and progression. Although traditional monolayer cell culture models have significantly enhanced our understanding of the disease, their results have not consistently translated into clinical success. These models lack the desired cellular diversity and fail to mimic in vivo physiological conditions. To address these limitations, our goal was to establish a human midbrain organoid model that closely replicates in vivo cellular diversity, tissue cytoarchitecture, and physiology. Within this thesis, we introduce a robust novel method for deriving human midbrain organoids from both healthy individuals and PD patients induced pluripotent cells (hiPSCs). We characterized the cellular diversity within these midbrain organoids using single-cell RNA sequencing (scRNA-seq), revealing the presence of dopaminergic neuron populations resembling in vivo substantia nigra neurons. We demonstrated the utility of the midbrain organoid model by capturing a comprehensive transcriptomic profile of dopaminergic neurons in response to SNCA triplication mutation and oxidative stress. The single-cell transcriptomics highlighted perturbations in oxidative phosphorylation and protein translation in response to SNCA-3X mutation. The vulnerability of dopaminergic neurons to oxidative stress was associated with the enrichment of cholesterol biosynthesis and synaptic signalling processes. Recent studies utilizing single-cell RNA sequencing have revealed that neuronal models derived from human induced pluripotent stem cells (iPSCs) using morphogens and small molecules exhibit undesired cellular heterogeneity and noticeable variability between different batches. Our aim is to develop a monolayer neuronal model that can offer highly homogeneous substantia nigra neurons on a large scale within a short timeframe through the ectopic expression of transcription factors (TFs). In this study, we validated a significant finding: ectopic expression of ATOH1 alone is sufficient to generate Tyrosine hydroxylase (TH)-positive neurons. Additionally, we identified several TF combinations that also induced TH-positive neurons. Single-cell RNA sequencing analysis of neuronal culture generated through ectopic expression of combination of (ATOH1 or NGN2), FOXA2, LMX1A, NURR1, PITX3, SOX6, and MSX1 revealed three distinct TH-positive neuronal clusters. However, these neurons were also found to be positive for PRPH, a peripheral nervous system marker. The characterization of TH-positive neurons induced by ATOH1 alone, with or without midbrain patterning factors such as SHH and FGF8b, also showed positivity for PRPH. Further refinement of the TF combination is needed to correct the regional identity of the TH-positive neurons. Additionally, the generated neurons require characterization beyond scRNA-seq/qPCR techniques. Human post-mortem material remains the gold standard for formulating hypotheses and subsequently testing them in both in vivo and in vitro models of PD. It also serves as an invaluable tool to validate hypotheses derived from experimental models of PD, confirming their relevance to human disease. In this study, we generated a comprehensive single-nucleus RNA sequencing dataset of a prominent PD-affected region of the midbrain, the substantia nigra pars compacta, from a large cohort of 15 sporadic PD patients and 14 control individuals. This study involved transcriptomic-level characterization of all major cell types, including their subpopulations, present in the brain. We demonstrated specificity of GWAS PD associated genes to different cell types. Importantly, we identified glial subpopulations enriched in the TH gene, which was found to be depleted in PD samples. This dataset provides a valuable resource for future hypothesis-driven experiment and represents a significant step forward in understanding PD's disease mechanisms.Item Embargo Relationship of brain functional, structural and vascular patterns to cognition in ageing and dementiaLiu, Xulin; Liu, Xulin [0000-0002-8219-2848]Cognitive decline with neurodegeneration is a major healthcare burden. To tackle this burden, it is necessary to better understand the brain-cognition relationship in ageing and neurodegenerative diseases especially at an earlier stage, ideally before cognitive impairment. Measurements from neuroimaging can be useful to identify the complex processes of neurodegeneration which involve structural, cerebrovascular and functional changes. In this dissertation, I investigated the following questions: 1) Can we explain the relationship between brain architecture and cognition by the integration, or dissociation, of structural connectivity and functional connectivity? 2) Do the patterns of functional network topography, brain structure and cerebral blood flow have independent or synergistic effects on cognitive function in healthy ageing? 3) Do the patterns of functional network topography, brain structure and cerebral blood flow have independent or synergistic effects on cognitive function at the presymptomatic stage of dementia? To address question 1, I investigated whether the increased reliance on maintaining functional connectivity for good cognition in old age is facilitated by structural network connectivity or independent of structural network connectivity. I used multivariate integrative approaches to assess the relationship to cognition across the adult lifespan of (i) shared signals between structural and functional connectivity and (ii) unique signals in functional connectivity that are independent of differences in structural connectivity. I found that the maintenance of cognitive functions in older people depends on functional connectivity supported by strong structural connectivity. This dependency was related to education level. To address questions 2 and 3, I combined multimodal neuroimaging to investigate the differences associated with cognitive decline across the adult lifespan and in genetic frontotemporal dementia. On healthy ageing subjects from the Cam-CAN cohort, I applied linked independent component analysis which allows for simultaneous characterization of MRI-based structural, cerebrovascular, and functional measurements. The purpose was to integrate all of these neuroimaging signals to better understand their contribution to cognitive decline in ageing. I then applied the same method to presymptomatic carriers of genetic mutations associated with frontotemporal dementia using the GENFI cohort. I found that among these brain patterns, functional network integrity, particularly the frontoparietal network integrity, was the most sensitive to the effects of genetic mutations and significantly correlated with cognitive performance both in healthy ageing and presymptomatic frontotemporal dementia mutation carriers. Together the findings suggest that brain structural, cerebrovascular and functional networks show cognition-related alternations while the individuals are still cognitively normal. This process can be affected by age, genetic mutation, education and the interplay between patterns within the brain. Functional network integrity and connectivity are particularly important for the maintenance of normal cognition, especially in older subjects and in those approaching the likely year of dementia onset. Integrating multiple neuroimaging measurements could be a promising approach to stratify phenotypic and genotypic heterogeneity in otherwise-fuzzy cognitive variability in ageing and neurodegenerative diseases.Item Open Access The role of Toll-like receptors in Parkinson's diseaseKennedy, CatherineInflammation in the brain has been strongly implicated in the development and progression of Parkinson’s disease (PD). Neuroinflammation in PD is thought to occur in response to aberrant α-synuclein aggregates, and may be mediated by Toll-like receptors (TLRs). TLRs are pattern recognition receptors with the ability to detect damage-associated molecular patterns. The activation of TLRs, potentially occurring through the detection of aggregated α-synuclein, triggers the release of pro-inflammatory cytokines. This may result in chronic inflammation, which produces an environment toxic to neurons and thus causes neurodegeneration such as that seen in the substantia nigra of the PD brain. I hypothesise that the blockade of TLRs will result in the slowing of pathological progression in an animal model, and the blockade of an α-synuclein inflammatory response in human PBMCs. Chapter 1 describes the clinical and pathological characteristics of PD and some of the suggested mechanisms behind the progression of pathology, focusing on neuroinflammation. Additionally, it introduces TLRs and summarises the current literature surrounding their connection to PD, in particular that of TLRs 2 and 4. Chapter 2 describes the reproduction of the Kuan et al., (2019) α-synuclein animal model, including behavioural and neuropathological features. To test the effect of TLR blockade on the development of pathology the drug candesartan was used. This is a licensed drug used in the treatment of hypertension through its action as an AT1 receptor blocker, but has also been identified as decreasing the expression of TLRs 2 and 4. This work demonstrated a protective effect of TLR blockade on the development of cholinergic neurodegeneration and olfactory deficits, both features of early human PD. Chapter 3 investigates the mechanism behind the protective effects of candesartan seen in chapter 2. Candesartan showed only small trends towards decreasing the expression and protein levels of TLRs 2 and 4. Neuroinflammation was not identified within the brain, and thus candesartan did not appear to have any effects on this inflammation. Peripheral inflammation was identified at a 2-month timepoint, and candesartan decreased this inflammation, providing a potential protective mechanism. Chapter 4 describes the optimisation of an *in vitro* assay to investigate the inflammatory response of human peripheral immune cells to different forms of α-synuclein. This work showed that human peripheral immune cells show a pro-inflammatory response to both monomeric and oligomeric α-synuclein, and that this is blocked using candesartan. This response was shown to be TLR4- but not TLR2-dependent. Chapter 5 summarises the main conclusions of my thesis. Candesartan is having a protective effect on the development of neurodegeneration within the α-synuclein animal model, however the mechanisms behind this are unclear. This protective effect could arise through the suppression of peripheral inflammation, through the inhibition of autophagy through decreasing the expression of TLRs on neurons, or through the blockade of AT1 receptors. Further evidence supporting an anti-inflammatory mechanism arises from the decrease in α-synuclein-mediated inflammation in *in vitro* human peripheral immune cells on candesartan treatment. This thesis finishes with suggestions for further work to provide more insight into the mechanisms behind the protective action of candesartan, and to provide further rationale for the use of candesartan in a clinical trial.Item Open Access Disease mechanisms and markers of progression in cerebral small vessel diseaseBrown, Robin; Brown, Robin [0000-0003-0431-7841]Introduction
Cerebral small vessel disease (SVD) is a common disease process accounting for a quarter of all ischaemic strokes, around 80% of haemorrhagic strokes, and is the major contributor to vascular cognitive impairment and dementia. Despite it being a major public health burden, understanding of the natural history is incomplete and the specific pathophysiological processes involved have not been fully elucidated. Consequently, there are few effective disease modifying treatments. In part I of this thesis I aimed to clarify elements of the natural history of white matter hyperintensity lesions (WMHs) in SVD. These are a key radiological feature of SVD that are strongly correlated with clinical sequelae, and I further tested whether brain lesion volume can regress over time. In part II I investigated the role of two novel pathophysiological mechanisms (inflammation and the permeability of the blood-brain barrier), and their relationship with SVD severity and progression. Methods
I performed a systematic review of WMH growth and used inverse variance-weighted meta-analysis to determine the expected WMH change over time in high-risk populations. I next used a novel timepoint-blind WMH marking technique to assess whether WMH volume regresses over time in three separate SVD cohorts. Finally I studied a cohort of patients with SVD undergoing PET-MRI imaging, phlebotomy and neuropsychometric testing. Results
WMHs typically expand at 2.50 ± 3.02 cc/year in patients with SVD and this is significantly more likely in patients with hypertension and who currently smoke. I found only 12/417 participants (2.9%) who showed modest WMH regression on longitudinal imaging, and this was more likely in patients with less severe disease at baseline. I demonstrated significant differences between patient and control groups in both microglial signal and blood-brain barrier permeability, and associations between microglial signal and both clinical and radiological markers of SVD severity. These disease processes did not predict disease progression at one year. Conclusions
I calculated the expected rate of WMH growth in relevant populations and how these data affect the sample sizes required to show a treatment effect, which should inform future trials. My results investigating WMH regression suggest that this is unlikely to be a significant factor in severe SVD. I showed that both microglial signal and blood-brain barrier permeability are likely to be relevant in SVD, but whether they are disease causing remains unclear. I further discussed the ongoing interventional study in which the data collection for this thesis was nested (MINERVA). The MINERVA trial aims to answer this question by testing whether minocycline can inhibit activated microglia and stabilise the blood-brain barrier, and I presented baseline data from the trial.Item Open Access Mitochondrial Dysfunction and Stress Responses in CHCHD10 Myopathy and NeurodegenerationShammas, MarioIn the last decade, dominant mutations in the mitochondrial protein CHCHD10 and its paralogue CHCHD2 were shown to cause familial amyotrophic lateral sclerosis and Parkinson’s disease, respectively, with phenotypes that often resemble the idiopathic forms of the diseases. Different mutations in CHCHD10 cause additional neuromuscular disorders, including the lower motor neuron disease spinal muscular atrophy Jokela type and autosomal dominant isolated mitochondrial myopathy. Modelling these disorders is revealing how mitochondrial dysfunction contributes to the aetiology of neuromuscular and neurodegenerative diseases. In this dissertation I generate and characterise a knockin mouse model of the CHCHD10 p.G58R mutation, which in humans causes a mitochondrial myopathy and cardiomyopathy. The G58R mouse recapitulates the human phenotype, developing a severe myopathy from birth and a cardiomyopathy later in life. I found that mutant CHCHD10 forms aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane as evidenced by prominent dilations of cristae containing membranous inclusions. Unexpectedly, the survival of CHCHD10-KI mice depends on a protective stress response mediated by the mitochondrial metalloendopeptidase OMA1. The OMA1 stress response acts both locally within mitochondria, causing mitochondrial fragmentation, and signals outside the mitochondria, activating the integrated stress response through cleavage of DAP3-binding cell death enhancer 1 (DELE1). I additionally identify an isoform switch in the terminal complex of the electron transport chain as a component of this response. Furthermore, I perform ultra-high depth sequencing of mitochondrial DNA (mtDNA) of CHCHD10 mutant mice and show that mtDNA deletion levels are higher in affected tissue, and the accumulation of these deletions happens in an age-dependent manner. I finally show that in addition to accelerating the rate of naturally occurring deletions, CHCHD10 mutations also lead to the accumulation of a novel set of deletions characterised by shorter direct repeats flanking the deletion breakpoints. I therefore here demonstrate that CHCHD10 mutations cause protein aggregation, inner membrane instability and mtDNA deletions with a distinct signature. This leads to the activation of an OMA1 stress response which is critical for neonatal survival, coordinating local and global stress responses to reshape the mitochondrial network and proteome.Item Open Access Imaging Correlates of Heterogeneity in the Syndromes Associated with Frontotemporal Lobar DegenerationWhiteside, David; Whiteside, David [0000-0002-5890-9220]The syndromes associated with frontotemporal dementia are heterogeneous in their presentation and progression, with variable correlation between clinical phenotype and underlying proteinopathy. Single pathologies are associated with diverse clinical presentations, while the same clinical presentation can be caused by multiple pathological entities. Heterogeneity makes predicting underlying pathology and longitudinal outcomes challenging in clinical practice and in research settings. I propose that a multi-modal imaging approach, including structural and task-free functional magnetic resonance imaging, will provide mechanistic insight into how phenotypic variance arises and improve predictions of disease progression and survival. In this thesis I draw from data for participants recruited at the University of Cambridge and from two multi-site collaborations, the Progressive Supranuclear Palsy Corticobasal Syndrome Multiple System Atrophy Longitudinal Study UK (PROSPECT-M-UK) and the Genetic Frontotemporal Dementia Initiative (GENFI). I describe characteristic differences in markers derived from task-free functional MRI and their relationship to patients’ clinical manifestations. I relate these functional changes to imaging markers of neuronal loss, cell death and synaptic loss. I find that subcortical atrophy from structural MRI relates to cortical functional network disruption, and that synaptic loss measured through [11C]UCB-J positron emission tomography affects behaviour in relation to changes in functional connectivity. I investigate differences in functional connectivity across the disease course. In individuals with familial frontotemporal dementia, time-varying functional network abnormalities predict symptomatic conversion in presymptomatic mutation carriers and future cognitive decline in symptomatic participants. In progressive supranuclear palsy and corticobasal syndrome between-network connectivity explains variability in survival but does not improve predictive accuracy beyond clinical and structural imaging metrics. Imaging-derived biomarkers in frontotemporal lobar degeneration need to be appropriately targeted at components of the neurodegenerative cascade. Task-free functional MRI is an objective and scalable neural marker of clinical syndrome, useful in detecting symptomatic onset and prognostication but limited by small effect sizes, poor signal-to-noise ratio, and moderate reliability. I discuss developments required in image acquisition and analysis to support clinical practice and trials of experimental treatments.Item Open Access Investigating protrudin’s role in neuroprotection and axon regeneration in the central nervous systemLove, Fiona; Love, Fiona [0000-0002-7805-5234]Protrudin — a transmembrane scaffold protein found in tubular regions of the endoplasmic reticulum (ER) — has previously been shown to strongly promote neuronal survival and axon regeneration after central nervous system (CNS) injury. This is in part due to increased levels of integrins in the distal axon, but this mechanism does not fully account for its beneficial effects. We have investigated protrudin's effects on intracellular transport, morphology, and protein localisation in neurons, and found varied but specific effects on different cellular systems. In particular, protrudin does not have any effect on the transport of late endosomes in CNS neurons — despite evidence for this mechanism in other cell types — due to the absence of key adaptor protein FYCO1 in mature neurons. It also does not have any substantial effect on dendritic spine morphology, so it does not indiscriminately promote cellular outgrowth. On the other hand, protrudin does interact with ER export and associated secretory machinery. Overexpression of an active mutant of protrudin increases the amount of an ER-Golgi intermediate compartment in axon terminals, and affects the transport of Golgi satellite organelles, which we observed even in the distal axon. Our data demonstrates that protrudin provides axons with the machinery for local membrane protein synthesis, which may play a role in neuron survival and regeneration. This work opens up new avenues for future research into adult CNS repair.Item Restricted